Bacteria; Proteobacteria; Alphaproteobacteria; Rhodospirillales; 'Acetobacteraceae'; Gluconacetobacter
Gluconacetobacter diazotrophicus is a symbiotic, plant-growth promoting bacteria. It was isolated from the roots and stems of Brazilian sugarcane plants in 1988 . Upon discovery, the bacterium was named Saccharobacter nitrocaptans. Due to its acetic acid production and similarity to previously classified bacteria, however, it was later renamed to Acetobacter diazotrophicus. Completion of 16S ribosomal RNA analysis led to a reclassification to its current designation and taxonomy [1,2,3].
G. diazotrophicus is a Gram-negative, nonspore forming and nitrogen fixing obligate aerobe . The temperature and pH growth optimums are 30°C and 5.5 respectively. The bacterium is acid-tolerant and can also both grow and fix nitrogen at pH of 3.0 and below [1,2]. Additionally, G. diazotrophicus grows optimally at a sucrose concentration of 10%, as found in its natural host, but is capable of growth at up to 30% sucrose under laboratory conditions. The bacteria has been shown to grow abundantly on other carbon substrates like D-galactose, D-fructose, and D-mannose .
Unlike many other bacteria that engage in symbiosis with plants, G. diazotrophicus is an endophyte and does not stimulate the production of nodules . Without a host plant, the bacteria will not survive in the soil for more than two days . Most host plants of G. diazotrophicus contain relatively high levels of sucrose, similar to the sugarcane on which it was discovered .
The ability of G. diazotrophicus to fix nitrogen and effectively promote the growth of its host plant opens the possibility for agricultural applications. Additionally, G. diazotrophicus has many other attractive characteristics. The bacteria is of monocot origin, less plant specific than other symbiotic nitrogen fixing bacteria, and does not require nodule structures for growth and nitrogen fixation . Given these factors, G. diazotrophicus could be a less costly and more environmentally friendly alternative to nitrogen fertilizers that the agricultural industry currently relies on heavily [6,7]. The bacteria could be adapted to colonize other monocot plants if sucrose levels were not a limiting factor. Monocot staples in agriculture include corn, wheat, and rice. These crops account for approximately 70% of the total world crop production . If nitrogen fertilizers could be supplemented or replaced by G. diazotrophicus colonization in these crops, that could lead to more sustainable and less environmentally damaging agricultural practices on a large scale .
G. diazotrophicus contains the Pal5 genome consisting of one circular chromosome (3,944,163 bp) and a G-C content of 66.19%. Additionally there are two plasmids present, pGDO1 (38,818bp) and pGDO2 (16,610bp). The genome was found to contain 3864 coding sequences. There were 1077 potential proteins sequenced in the genome, with 583 being identified to the metabolic pathways within G. diazotrophicus. Studies have shown that expression is dependent on the crop present (such as sugar cane) and the levels of nitrogen present in the environment. Genes were found to augment resistance to acetic acid, which allows G. diazotrophicus to fix nitrogen in very acidic environments. 
Cell Structure, Metabolism and Life Cycle
The bacterium’s cells are shaped like straight rods with rounded ends and motility is provided by 1-3 lateral or peritrichous flagella. Cellular dimensions are approximately 0.7-0.9 μm x 2 μm . When viewed under a microscope, cells are single, paired, or chainlike in structure.
G. diazotrophicus reduces atmospheric nitrogen to ammonium by a molybdenum (Mo)-dependent nitrogenase . Mo-dependent nitrogenase contains Fe protein and MoFe protein . The Fe protein is a homodimer that has a binding site for MgATP in each subunit. When the Fe protein is complexed with MoFe and two MgATP molecules, the MgATP is hydrolyzed to MgADP, and the MoFe protein binds the substrate for reduction . The bacteria’s lack of a nitrate reductase protein suggested that nitrogen fixation would not be inhibited by feedback from nitrogen assimilation, but nitrate still may inhibit the process [3,13]. Low levels of fertilizer do not inhibit nitrogen fixation, so G. diazotrophicus may be a useful supplement to ammonium based-fertilizers [11,14].
G. diazotrophicus is a chemoorganoheterotroph. Glycolysis and the pentose phosphate pathway are found in G. diazotrophicus . Its carbon sources include d-galactose, d-arabinose, d-fructose, and d-mannose . The main pathway for glucose oxidation occurs in the periplasm. Pyrroloquinoline quinone-linked glucose dehydrogenase (PQQ-GDH) oxidizes glucose to gluconate extracellularly [16, 17]. The enzyme is produced to meet the energy requirements during nitrogen fixation . G. diazotrophicus also uses nicotinamide adenine dinucleotide-linked glucose dehydrogenase (NAD-GDH) for intracellular glucose oxidation . Levansucrase, a fructosyltransferase exoenzyme, helps G. diazotrophicus to survive in high sucrose environments by aiding in sucrose transport . The enzyme acts extracellularly to hydrolyze sucrose to fructooligosaccharides and levan [19, 20]. The levan produced by the enzyme is also critical for biofilm formation by the bacteria [21, 22].
G. diazotrophicus can live and reproduce intracellularly or extracellularly within plants. Sugarcane produces anionic glycoproteins capable of binding G. diazotrophicus as a mechanism of selection for the endophyte . The bacteria are more likely to enter the plant at the root cap on root tips where there are loose cells. Entry through the stem occurs at openings where young plants have diverged. In leaves, G. diazotrophicus can enter a wounded stomata . Vector transmission by the sap eating pink sugarcane mealybug, Saccharococcus sacchari, is also possible . Production of bacterial hydrolytic enzymes assists in colonization [24,26]
Ecology and Symbiosis
G. diazotrophicus was initially isolated from sugarcane in Brazil but later found to be associated with sugarcane in Philippines, Japan, Argentina, Cuba, Mexico, Australia, Mauritius, and India . It is an endophyte that can colonize intracellular or intercellular spaces in sugarcane roots, leaves, and stems. In other crops like carrots, radish, beetroot, coffee, and rice, G. diazotrophicus has been isolated from the rhizosphere .
Plant Growth Promotion
Carbon sources from plants benefit the bacteria, but there are numerous ways G. diazotrophicus supports plant growth. Nitrogen fixation by G. diazotrophicus produces about half of the required nitrogen for crops [27,28]. The bacteria also promotes plant growth through phytohormone production. Production of indole-3-acetic acid and gibberellins promotes growth of the root systems and shoot systems in plants . G. diazotrophicus provides nutrients to plants by solubilization of phosphorus and zinc . Outside of growth promotion, G. diazotrophicus protects sugarcane from Xanthomonas albilineans, the causative agent of leaf scald, which results in wilting, necrosis, and plant death . Production of a Linocin M-18 like bacteriocin called gluconacin prevents leaf scald by lysing X. albilineans . The bacteriocin is highly conserved in the Actetobaceterceae. The bacteria’s antagonistic capabilities make it a candidate for biocontrol in agriculture. Potential uses of gluconacin include application of teh bacteriocin of gluconacin to plants and tools to prevent infection by X. albineans. Engineering of transgenic plants capable of producing gluconacin could further prevent leaf scald in crops .
G. diazotrophicus shares low 16s rRNA similarity with the two human pathogens within the Acetobacteraceae family. It has no history of infecting humans .
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Page authored by Isaac Coker, Kyra Colston, and Danielle DeCesaris, students of Prof. Jay Lennon at Indiana University.